Column chromatography

How to pack, load and run a chromatography column

[@bethg7026]

Hi everyone. I just discovered that KZbin is showing ads on my videos. Please know that I am not monetized, as I don't have enough subscribers, so I do not make any profit off of those ads. Skip away!

[@bethg7026]

Oh my goodness, in over 3 years I've never known that I mis-spoke there and said the least polar is the one that moves the most AND its the one that moves the least! Some day I'll pull this back onto my computer, throw in a correction and re-upload it!

[@Marqueee.z]

Hi! How much silica did you use?

[@bethg7026]

@Pamela Loise Marquez looks like 10mL from the beaker. Sorry, this was so long ago and I don't have the manual any more

[@victorvorel7535]

Very natural manner, well explained and quite cute !

[@RP-bg2os]

Thank you so much for such a clear explanation, didn't get bored for even a bit in a 45 min long video 👍👍

[@bethg7026]

Wow that is an amazing compliment, given how bored watching this entire thing makes me!

[@nadanada3960]

Very nice, simple and detailed explanation. Thank you!

[@bethg7026]

Thank you!

[@sjackson1739]

I finally understand this better. Thanks so much for this video!

[@bethg7026]

You're welcome, I'm glad it helped!

[@bethg7026]

Hi everyone, I am so surprised that there have been so many views on this video! If I were to start making these again, what type of video would you like me to make? Please bear in mind that I have changed jobs, I can't make videos in a lab with equipment any more, but I can absolutely make videos with drawings (like the intro to the TLC video).

[@siliconvalley3271]

Good video. In the test tube we got coloured compounds (along with eluent) . How ca we separate further eluent and compound .

[@abdullahe3674]

yes Prof, you an ocean of knowledge throw it at us , we thursty with much chemisty love kind regards :)

[@bethg7026]

@@abdullahe3674 that's so sweet, thank you! I think I will make a video this summer on how to study for a chemistry class

[@theghostofsw6276]

@@bethg7026 I'd like to see a video on vacuum sublimation. Lots of videos on the sublimation of caffeine...but none touch on the actual degrees of heat, amount of vacuum applied, and why. It's almost as if these things don't matter?

[@bethg7026]

@@theghostofsw6276 its not so much that it doesn't matter, it's that the heat and vacuum needed to perform a vacuum sublimation are actually related to each other. The higher the heat, the less vacuum needed. The videos may also be using a vacuum source in which they have to control over the pressure, as settable vacuum pumps are very expensive. Whether it's a vacuum sublimation or distillation, if you don't have the ability to control the pressure from your vacuum source then you just turn on the vacuum and heat your pot until it sublimes or distills. I could certainly make a video of the technique itself/ however it would be all hand-drawn or use open source pictures, as I no longer work in a chemistry department and therefore don't have access to the lab 😔

[@ggezwpgjnpgaming2186]

Great video, Ma'am! Thank you so much!

[@bethg7026]

Glad it helped you!

[@vrushbuskitchen1631]

hiii i m a MSc student and this video is very helpful for me...so thank u so much...

[@millardemmanuelbalatucan4947]

Thank you for this guide! really helpful during this virtual setup

[@bethg7026]

Thanks, I'm glad you found it helpful!

[@roryboyes2307]

I'm watching due to curiosity & do not have much chemistry knowledge & you made the topic very approachable. Thank you for your time.

[@bethg7026]

Aww that's so sweet! Comments like this make me feel that the 6+ hours that went in to making this video were worthwhile!

[@blessedgreta6669]

Hi Beth thank you so much for this detailed experiment. Always found the whole chromatography process as complex and difficult but you just made me thoroughly understand it. Obrigado❤️🥰

[@bethg7026]

That's awesome, I'm glad it helped you!

[@youthclub1122]

It's really amazing

[@leanswagger5822]

32:41 just to note little mistake, otherwise great video, thanks! on a TLC plate, the least polar compound is at the top and the MOST polar compound is at the bottom.

[@triskanelanda6572]

Hi Beth G. Thank you very much for making this video. It is very informative and detailed

[@bethg7026]

Thank you, I'm glad that you find it helpful!

[@DivineAsHarUnion1122]

Great video 👍🏻

[@naboria5]

Thanks you very much. Excellent experiment.

[@bethg7026]

Thank you Pedro, although I can't take credit for the experiment itself!

[@lilibeldelapuente2163]

Love your candor in "not an organic chemist, terrified." I'm also a chemist trying to learn more chromatography.

[@bethg7026]

Hahaha I was SO nervous...the last column I had run was back when I was taking undergraduate organic chemistry, way back in 2000. And I've not run once since that video either!

[@lilibeldelapuente2163]

@@bethg7026 You killed it! I did the dye mix today but I used Brilliant cresyl blue, Methylene blue and neutral red. I got a very clear red/blue separation but not two blue spots. I used isopropyl alcohol to dilute the dyes, dotted the plate, then placed in the chamber with 1:1 acetone:water. Any idea what eluent I could use for separating the blues?

[@bethg7026]

@@lilibeldelapuente2163 I honestly don't know, but you can try using TLC to play around with the polarity of your solvent mix. Figuring out the solvent mix is all about trying to get one to move faster than the other by making one like the solvent more than the silica.

[@rj23032]

Oh your voice is so pleasing Beth!

[@bethg7026]

Why thank you!

[@carloshenriquefurtado6900]

Parabéns! Excelente vídeo.

[@bethg7026]

Obrigado!

[@fakhrulnawawi9681]

Very clear instructions..thanks 🥰

[@bethg7026]

Thank you! 😁

[@adrianosantos1483]

Vídeo incrível! Muito obrigado! Você é uma professora incrível! Abraços do Brasil!

[@bethg7026]

Muito obrigado, fico feliz que você ache isso útil!

[@siliconvalley3271]

Good video. In the test tube we got coloured compounds (along with eluent) . How ca we separate further eluent and compound .

[@bethg7026]

@@siliconvalley3271 if your eluent has a lower boiling point than the compound, a rotary evaporator is the way to go. If the compound has the lower boiling point, distill it. If you dont have a rotary evaporator, you could distill the solvent away, but you need to make sure you dont overheat the pot, heat it just hot enough so that the solvent boils. Maybe use a water or oil bath to better control the temperature.

[@nimrahtahir8474]

Thank you so much for this informative video....😍

[@bethg7026]

Thank you Nimrah, I'm glad you found it helpful!

[@saniasohail731]

thanknyou for this video

[@NathanaelNewton]

I love your goggles.. I'll have to figure out where i can get some :D

[@bethg7026]

They are Uvex Stealth, and very comfortable! Try to find the elastic strap version, the neoprene strap gets brittle and breaks in under a year.

[@victorprokop9343]

great content

[@bethg7026]

Thank you!

[@hazellevesque692]

Hello. First of all, thank you so much for the amazing video. Also I really appreciated that you answered everyone you could in the comments, even the new ones despite how long it has been since you published the video, it's truly admirable. I had a question of my own too: So, I am a trying to write a paper on the relationship between viscosity and separation efficiency in gel permeation chromatography. It's actually a physics paper, so I'm trying to focus on the fluid dynamics of it mostly. I was going to try to change the solvent I use for the silica gel to explore different viscosities, but would it cause an imbalance in the experiment by also changing its polarity? Is there a safer or clearer way to explore this topic without getting into the chemistry of it too much, or do you have any different recommendations? Sorry if my question is too long or not clear, but I'd really appreciate if you could help. (PS: I am planning on separating a food dye into its components, as you did.) All the best!

[@bethg7026]

Thank you so much for your kind compliments, I really love to see how many people are still using this horribly long and boring video to learn! I think your question has a couple of different points to consider, but be forewarned that I know very little about size exclusion chromatography. First, if you change the polarity of rhe solvent, would those change the way in which it interacts with the polymer chains? If it does, could this change the size of the coils? If it does, I think that might change the retention times because they won't permeate the beads in the same way. Second, if you do have a very large polarity change, will this have an effect on the stationary phase itself? If it irreversibly bonds to the stationary phase, will it effectively decrease the pore size? If so, I would think that could also affect retention times. I thinknyour safest bet would be to stick with eluents that have similar polarities but different viscosity. And then you could maybe do a second paper looking at same viscosity but different polarity 😉

[@phamquocvietvuong8137]

thank you so much...

[@bethg7026]

You are more than welcome 😀

[@iqrahoor5238]

thank you

[@bethg7026]

You're welcome!

[@ihtsarl9115]

Thank you for this video so silica gel will absorb the acetone leaving only ethyl alcohol to drip down is that it?

[@bethg7026]

Not really, it's more that the compound has a higher affinity for the eluent than it does for the silica. The silica doesn't absorb anything either, it absorbs (compounds stick to the outside of it instead of going inside).

[@the_harlekin]

Thank you for this video, it's great! I have one question: What are you donig with the sand and the silica gel after the chromatography? Is it possible to seperate them from each other and use it again? Or do you have to dispose it?

[@bethg7026]

Thank you, I'm glad you liked it,! As much as I love sustainability, this is a one-time use material. As this is a purification technique, it's essential that the silica and sand are both very clean, and there is no good way to wash them.

[@shelbysgarage5828]

Hi Beth, I'm a hobbyist and teaching myself how to do flash chromatography. The dyes you used. Am I correct thinking you started with a mix of 3 different dyes? And is it fairly standard to add dyes? Is there a relationship between the choose dye and a compound your trying to purify? Like say methyl blue and (insert compound) would stick together better due to polarity? And due to eleunt?

[@bethg7026]

Hi there! Yes, this was a mixture of 3 "dyes" (actually acid-base indicators), dissolved in ethanol. This was just the solution mixture I used to show the different components of a mixture separating as you run the column. We do NOT add dyes, or anything else, to the column. Doing so could cause a chemical reaction to occur, which would change the polarity of the compounds you are trying to separate. Chromatography is just a way purify a compound that is dissolved in a solvent by separating it from the other solutes, no chemical or physical changes should ever be undertaken during chromatography.

[@metiscus]

They normally use two or three dyes when you are learning so you can see the separation. Most solutions you are separating aren't colored so you will use TLC against a standard to determine the order, or sometimes you'll use spectroscopy. When working "blind" that is without color, you take timed fractions and check those using either TLC or spectroscopy etc. In HPLC, that testing is basically automatic and the column is usually under pressure to speed it up as well.

[@blackandwhite252]

what could a dried out column do to the elution and isolation of pigments?

[@bethg7026]

Sorry for the late reply, I totally missed that you had asked a question! When you allow a column bed to dry out, two things happen. First, the silica and your compound losing the solvent basically restarts the elution process, so you restart the separation from wherever your band is on the column. It's not just a pause, it's a complete restart. So you can just add more solvent, the air goes away and you're good right? Nope! You're not going to be able to just add more solvent and get rid of that air, some of it will stay in the silica and the bed will also form cracks that you can't rid of. Wherever an air gap stays, your stuff won't move down the column. If you're lucky it will to the side as solvent flows...but you need everything to run straight down, not to the side, or you ruin that nice thin band you were hoping for. End result is, even with a good solvent mix that would give you nice, thin, perfectly separated bands, you end up with streaky, thick bands that may end up overlapping, nothing comes of where it should, etc.

[@tulsilimbu7671]

❤❤❤

[@malchikscott]

Hi, Beth. Loved this video! I'm writing a novel with a scene where a high school student uses column chromatography to separate toxins in copperhead snake venom. He's looking for a cure for cancer. Your video was very helpful. I'd love to run some of my character's homemade, jury-rigged science experiments by you. Would you be interested in reading them and giving me feedback?

[@bethg7026]

I'm glad that you liked this video! I would be more than happy to, can you dm me on twitter (@bethg79)?

[@parthapanda4455]

Please Beth provide the lab manual. I will be very grateful if you provide the labmannual in the description

[@bethg7026]

I'm sorry, I can't do that as the manual is copywritten material, by Christina Goudreau Collison at RIT.

[@parthapanda4455]

@@bethg7026 it's okay 😄 the demonstration is still one of the best and enough explaining.

[@bethg7026]

@@parthapanda4455 thank you so much!

[@clashwithsparkcrazygamers]

Is there a specific amount of silica you used for the preparation of this slurry ?

[@bethg7026]

There was but I don't remember how much I used! Its really a balance between how much product you are running through the column and how effective your eluents are at separating your compounds. A good rule of thumb is 1:30 (30g silica per 1g of compound) for gravity columns.

[@salehchem4376]

thank you so much. Is there standard to use silica gel in column, I mean if my sample a little bit like (0.01 gram), how much of silica gel use here? in contrast, if I have a lot of samples such as (1 gram or more), here, how much of silica gel have to use? I mean is there ratio from silica gel to sample to use in column, please tell me

[@bethg7026]

General rule of thumb is 30g silica for every 1g of sample. When I was making .1g batches of product in graduate school I used a pasteur pipet as my column.

[@salehchem4376]

@@bethg7026 ok thank you so much

[@sh7arwt3teer99]

please if you can explain to me, why the mobile phases containing a concentration of ammonium sulfate promote the binding of many proteins to HIC columns??

[@bethg7026]

Sure! Ammonium sulfate in the mobile phase interacts with the protein to have it expose more of its hydrophobic areas, which are what stick to the stationary phase.

[@Leen-ih8ql]

@@bethg7026 Thank you so much

[@fakhrulnawawi9681]

5:20 what solvent was that? Is it the mobile phase etoh hexane 1: 1 or absolute hexane?

[@bethg7026]

I'm sorry, I don't remember. This was 4 years ago, and I no longer work jn the department. It was a mixture of cyclohexane and something else. The eluent is going to completely depend on what compounds you are trying to separate anyway, so what worked for my demonstration would most likely not work for your mixture anyway.

[@youthclub1122]

Each step that u do is Soo important I just want to take a notes can u z snd me ma procedures

[@bethg7026]

Hi there! I'm glad you liked my video. Unfortunately this experiment was protected by copywrite, even if I did have it I could be sued if I shared it.

[@youthclub1122]

@@bethg7026 no problem ♥️

[@dedeagustinaa]

I have any question mom, can i change Sand in the top with drysilica?

[@bethg7026]

No, this will just add more silica, and possibly air bubbles, to the column. You don't have to add the sand but it protects the top of the silica column from being disrupted when you add your eluent, which will mess up how nearly your bands elute.

[@salehchem4376]

If I used wet packing of silica gel to column (slurry), here, can I use any solvent for silica gel until reached slurried, or must use only hexane. thank you

[@bethg7026]

Now that I've not even thought about columns other than to answer these videos, I'm a little vague on the details, but if I remember correctly you typically pack it with the same solvent mixture you intend to run through it first.

[@salehchem4376]

@@bethg7026 ok thank you

[@paryjaafari4253]

Hi everyone, is there any relationship between the maximum volume of chromatography column and our impure mixture?

[@bethg7026]

Absolutely! First, you need to make sure that the amount of silica gel is appropriate for the amount of sample, and the ability of your solvent system to separate the components. Then, you need to make sure the column is wide enough to allow you to load your sample as a thin band. Lastly, you want the column to be long enough so that you've filled at least 2/3 of it. A more detailed guide with actual numbers to follow is available at www.chemistryviews.org/details/education/2101817/Tips_and_Tricks_for_the_Lab_Column_Choices.html

[@nicole7323]

Thank you a lot!! 🙌🏼👏🏻👏🏻 excellent explanation!

[@bethg7026]

Thank you so much! It amazes me that this video is still helping people all these years later!

[@joramforbes7923]

in the experiment of column chromatography of plant pigments (we used spinach), what would happen to the color bands if acetone and not hexane was used as the first eluting agent?

[@bethg7026]

Given the timing of this question, I'm going to assume it's from a test. Think about what the effect is of solvent polarity - does a more polar solvent make things elute faster or slower? Then, figure out which one is more polar, acetone or hexane.

[@joramforbes7923]

@@bethg7026 so basically, qualitatively- the colors would not have any effect on the said condition? it's just about the eluents' polarity? i was just confused with the given lab question

[@bethg7026]

@@joramforbes7923 the colors are the components you are trying to separate. The speed and separation in which they run are dictated by the polarity of the solvent you used to run the column. More polar solvent will either make them run through faster and closer together, or make them run slower with greater separation (or not move from the top of the column at all). You need to figure out which one is the case.

[@bethg7026]

@@joramforbes7923 so what was your final answer?

[@bethg7026]

@@joramforbes7923 couple things... 1) there is absolutely no chemical reaction going on here. Chromatography is just a way to separate chemicals in a mixture (in your case, the chemicals that make up the color in spinach). Acetone doesn't react with or consume hexane. It does make things elute faster because it's more polar. 2) it won't make the bands more evident, and I don't understand why you say they won't be "disturbed". The rate at which the bands move through the column will change, that's the point of changing solvents. I recommend you watch my video on TLC (note, I make money on my videos, I'm not trying to earn anything by sending you there) This goes into more detail on the effect of solvent. TLC and column chromatography are essentially the same thing, in TLC the silica is just mounted on a plate and the stuff elutes from bottom to top, vs a column that has a tube of silica and elutes from top to bottom.

[@salehchem4376]

happy new year. If I have solid sample containing components, can I use any solvent to dissolve it? Or I have to use only the same mobile phase as solvent to dissolved sample? Another case, if must use same mobile phase, but my sample can not dissolve, here, please I need solution to this problem. can I use any solvent? thank you alot

[@bethg7026]

If its not soluble in your selected mobile phaseyou need to change mobile phases. Your compound has to be soluble or it won't move down the column...

[@salehchem4376]

@@bethg7026 thank you

[@vaibhavipatil572]

Mam can u plz tell ....which solvent u use to put the sand at bottom

[@bethg7026]

The sand is dry, but if i rinsed any of it down it was thw firat solvent mix that i was going to run through the column (which js what you mix with the silica gel with to pour it into the column too)

[@ninjabrozgaming6053]

how can we select mobile phase for the isolation of new compound?

[@bethg7026]

Hi Shweta, what you do is test solvents by running TLC, until you have a good separation. It can be a little more complicated than this (you often have to cut the polar constituent way down when you switch from TLC to a column and you may need to see if the compound will degrade during elution), but you always test and tweak on TLC before you run a column.

[@ninjabrozgaming6053]

@@bethg7026 hello mam I used chloroform, methanol and water and got the 4 separate spots on my tlc plate. So can I use this mobile phase for column

[@bethg7026]

@@ninjabrozgaming6053 that's a really difficult question to answer on here. I would suggest trying it out using a small amount in a micro column (make a column in a pasteur pipet). If it works there it should be ok to scale it up to a full size column. If you don't get good separation, tweak your ratios to get better results (often you have to decrease the amount of polar solvent when you go to a column because all of your stuff elutes too quickly) I need to explain to you that I am absolutely not an organic chemist, I'm about the worst person to ask for advice on the finer details of selecting mobile phases! I would highly recommend you look at some other videos specifically about finding a mobile phase, or head to your library to find a book about column chromatography.

[@ninjabrozgaming6053]

@@bethg7026 OK thanks mam

[@Luke-ii2qb]

Hi Bet, I'm going to chromatograph Cannabinoids. What solvent do you recommend? I am testing with methanol and ethanol. To evaporate them later. I would like to separate the THC from the CBD. Thank you so much!! your videos are awesome!!

[@bethg7026]

A couple words of caution here...first, don't be a home-based drug scientist. Just don't. Second, don't buy chemicals, solvents or materials that are not pharmaceutical-grade and use them on something you intend to ingest/inhale/inject/put on your skin. Third, don't mix your stuff with something toxic unless you are 100% certain that you can get every last trace of that toxic substance out. Simple evaporation is not guaranteed to get rid of 100% of the methanol, as it may bind to the product, form an azeoptrope, etc. Basically...don't go there. Please, for your own safety.

[@youthclub1122]

Can u plz send me a procedure in a written foram

[@pallavichampaneria961]

Mam pls explain how can we seperate colourless compound with the help of column chromatography?

[@bethg7026]

Assuming the compounds will show up on a TLC plate, you use TLC as your guide. First you use TLC to determine the eluent mixture to use. Then, you run the column as normal, collecting small portions of what comes out in individual test tubes, and spot each tube on a piece of TLC plate to see which ones have your compound in it.

[@galiparthijoshna6155]

Hi everyone. I am having a doubt that instead of using silica gel I found that we can use alumina but in which solvents it can be soluable . Pls anyone reply me .

[@bethg7026]

Hi there! In terms of solubility of alumina, you've nothing to worry about, it's not soluble in anything that i know of except concentrated strong base (KOH). It will not dissolve in water or organic solvents Now for the cautions. You can use alumina, but it's not something that you can just swap in and expect the same eluent to work. Alumina is more polar, preferentially binds acidic compounds (silica does this to bases, and in fact most amine separations are done with alumina because they like to bind too strongly to silica), and comes in different pH's (acidic, neutral and basic) and different water content (type I, II or III). It's not as easy as the "this jar of silica will work for pretty much anything", and we don't generally start with it because it can take so much time an wasted product to figure out how to make alumina work. Usually, if you can separate using silica, you go for silica. It's just easier. Alumina is usually used if silica fails, which is often when you have basic compounds, compounds that are not acid-stable (silica is slightly acidic and will destroy those), or your compounds are so similar in structure that you need the enhanced polarity of alumina to get better separation. There are likely lots of internet pages that go into good detail on this, I would suggest looking up "alumina vs silica" to get more details to help you!

[@biologicalmints9510]

which (solvent) name the solvent u add after cotton

[@bethg7026]

I honestly dont remember what my eluent was, but you use whatever eluent mixture you are going to start with.

[@biologicalmints9510]

@@bethg7026 thank u 😊

[@ahmadomega9253]

Hello, Mrs. Beth G, i want to asking about size of column you using in this video, can you describe to me? Thanks

[@bethg7026]

Oof it's been a while. I want to say it's a 1.5cm diameter column. I bought those almost 10 years ago!

[@ahmadomega9253]

@@bethg7026 thanks for information Mrs. Beth

[@madman026]

to think she is unknowingly teaching you how to make risen poison as well:)

[@bethg7026]

Fyi, this was posted for our organic chemistry courses as a way to teach them about column chromatography instead of making them short through a boring prelab lecture

[@madman026]

@@bethg7026 aye :) just going to the fact if you figure out how chromatography works a lot of poisons can be made

[@bethg7026]

@@madman026 technically no, since you're not making anything by running a column.

[@madman026]

@@bethg7026 ok ok i jumped ahead to the next step sue me

[@bethg7026]

@@madman026 lol obviously you are not a chemist...first you make it, then you purify it

[@ssangeetha6975]

What is the eluent name

[@bethg7026]

It was a mixture of two solvents, I honestly don't remember what they were.

[@tuhoang9569]

Hi, can I have information about the chromatography column?

[@bethg7026]

What information are you looking for?

[@tuhoang9569]

@@bethg7026 thanks, i would like to know the height and diameter of the chromatographic column

[@bethg7026]

@@tuhoang9569 oh boy, it's been a while since I made this video, and I've not been in that department in over 3 years now. I want to say that was a 1.5cm diameter column, and maybe 18" tall...it was way too tall for what I needed, but I couldn't get one any shorter.

[@jenstorp8086]

silica gel handled out of a fumehood? that is kind of a safety hazard

[@bethg7026]

Not when it isn't airborne, which it won't be when it's slurried into a solvent. When our students are ppuring it dry, its in a fume hood. I chose not to film anything inside hoods because of the background noise. Also, silicosis is a hazard upon chronic respiratory exposure, not acute. But, both for the protection of the instructors and for any students who are also being exposed to respirable silica elsewhere in their lives, dry silica is used in our fume hoods. The only time they pour it out of the hoods is when it's being poured as a slurry into the column.

[@nassimabk3714]

32:22

[@bethg7026]

Yes, that's where i say that the least polar comes off first...

[@nassimabk3714]

@@bethg7026 hhhhhhh yes. I have been mixing up between normal and reverse chromatography. I love your video! especially with the way we've been studying this has been the best thing ever..thank you!

[@bethg7026]

@@nassimabk3714 aww what wonderful praise, I'm glad that it helped you!!